The present invention relates to quantifying (or measuring the amounts) of reaction compounds which are fraction collected in microtiter plate format.
Systems exist for the parallel synthesis of various chemical compounds in microtiter plate format. Accordingly, many liquid handling systems have been developed for automated parallel synthesis in conjunction with such microtiter plate formats. High throughput liquid chromatography/mass spectrometry systems capable of isolating compounds on the basis of their molecular weight have also been developed. However, a system for easily and efficiently determining the total collected masses of compounds which are fraction collected in microtiter plate format has proven elusive.
In general, existing high throughput liquid chromatography/mass spectrometry systems isolate compounds by fractionation into fraction collector tubes which are individually removable from a rack. Numerous problems exist when attempting to quantify the total mass of each of the compounds collected in the various fraction collector tubes. First, each tube in the rack needs to be individually removed, weighed and its weight recorded. The compounds are then fraction collected into the individual tubes in the rack. Subsequent to fraction collection, the tubes are removed one after another from the rack and are placed in a device which concentrates them down by driving the contents of the tube. After the contents of the tube have been concentrated down by drying, the dried tubes are individually re-weighed and the net weight of each sample in the fraction collection tubes is then determined by comparing the initial weight of the empty tube with the weight of the tube with the dried compound therein. The weight information so determined allows the compounds to be redissolved with a volatile solvent to a desired set point molarity. The tubes are then placed into a rack and an aliquot is delivered into a microtiter plate for use in a biological assay.
In high throughput synthesis operations, the number of samples can be quite large so the operation of separately weighing, re-weighing, tracking, and labeling a very large number of fraction collector tubes becomes quite cumbersome. Moreover, when weighing and re-weighing relatively small volume fluid samples, inaccuracies in the weight of the tubes themselves may account for larger weight differences than the weight of the compound fraction collected therein. As such, significant errors can be introduced to the net weight of each collected compound, and its final concentration upon dissolution. In addition, special devices such as racks and an apparatus for concentrating down fluid samples have to be used with the dried down products later having to be reformatted into a microtiter plate format.
The present invention provides methods and systems for measuring the total masses of individual compounds present in fluid samples, and is particularly well adapted for use with fluid samples prepared in microtiter plate format. In a preferred aspect, the individual compounds are isolated by fluid separation systems including high performance liquid chromatography columns and supercritical fluid columns. Small portions of the individual compounds isolated by fluid separation are then analyzed by a mass spectrometer, (which determines the compound molecular weight), and by an evaporative light scattering detector, (which determines the total masses of each of the isolated compounds passing therethrough). The present invention provides a single pass system which measures the masses of the individual compounds diverted into fraction collectors in real-time, as follows.
The signal generated by the mass spectrometer is used to determine the interval of time during which fraction collection is to be carried out. The signal generated by the evaporative light scattering detector is used to determine the total amount of mass diverted therethrough during the interval of time during which fraction collection is carried out. By knowing the portion of fluid sample diverted into the evaporative light scattering detector, (relative to the portion of fluid sample which is directed to fraction collection), it is then possible to calculate the total masses of each of the individual isolated compounds as they are fraction collected.
By selecting intervals of time for fraction collection on the basis of when a desired compound is present in sufficient concentration, (as indicated by the signal strength from the mass spectrometer), the individual isolated compounds can be purified as they are fraction collected. Thus, an advantage of the present invention is that, during only a single pass through the system, each fluid sample is partitioned into isolated compounds which are individually weighed in real time as they pass into fraction collectors.
An additional advantage of the present invention is that microtiter plate formats can be used both for sample preparation reactions and for fraction collection of the individual compounds present in such samples. Accordingly, the present invention provides a single-pass system for the synthesis, isolation, weight measurement, and delivery of compounds in microtiter plate formats.
Another advantage of the present invention is that it is able to determine the total mass of the individual isolated compounds present in microtiter plate fluid samples at the same time that these individual compounds are being fraction collected in microtiter plate format. As such, only a single pass of a fluid sample through the system is required to isolate the individual compounds in the fluid sample, to fraction collect the individual isolated compounds and determine the total collected masses of these isolated compounds.
Therefore, an additional important advantage of the present microtiter plate format single pass system is that it completely avoids individually removable fraction collection tubes. Accordingly, it is not necessary to individually weigh individual tubes containing fractionated fluid samples. Consequently, it is not necessary to separately track and label such tubes or to transfer individual tubes between various liquid handling, concentrator and fraction collection devices. As such, the problem of individual tubes being misplaced or transposed and the amount of time involved with transferring individual collection tubes is completely avoided. Thus, another advantage of the present invention is that by using a high density microtiter plate format, the present invention reduces the actual amount of material which needs to be synthesized, (as compared to existing non-microtiter plate format systems), thereby resulting in cost savings through the reduced costs of synthetic chemicals. By using a small volume microtiter system, solvent consumption is reduced. Moreover, being a small volume system, significant reduction in the disposal of waste streams is achieved.
An additional advantage of the present small volume microtiter plate format is that, in preferred aspects, more difficult chromatographic separations can be achieved with the resolution of present invention""s preparatory or semi-preparatory chromatographic column where the resolution approaches that of a conventional smaller analytical chromatographic column.
Existing systems which remove and re-weigh fraction collection tubes individually can not be operated with the small fluid sample volumes of the present microtiter based system since inaccuracies in the weight of the tube itself may account for larger weight differences than the weight of the collected fluid sample therein. As such, significant errors can be introduced to the net weight of each product. The present invention completely avoids this problem.
In the present invention, a plurality of fluid samples are first provided in a standard microtiter plate format. An autosampler is then used to sequentially load the fluid samples onto a fluid separation system which preferably comprises a preparatory or semi-preparatory high performance liquid chromatography (HPLC) column or a supercritical fluid chromatography (SFC) column, which separates the components in each fluid sample such that individual compounds are isolated by sequential elution from the fluid column. Keeping within the scope of the present invention, however, alternate systems which facilitate fluid based separation can be used.
A very small portion, (typically on the order of 1%), of the sequentially eluted isolated compounds in the separated fluid sample are diverted into a mass spectrometer which determines the molecular weight of the individual isolated compounds passing therethrough. The signal generated by the mass spectrometer will correspond to the molecular weight of the compound passing therethrough such that fraction collection can be performed when a desired isolated compound is present in a desired concentration. As such, the output signal of the mass spectrometer can be used to signal the interval of time during which fraction collection is to be performed.
In addition, a very small portion, (typically on the order of 1%), of the sequentially eluted isolated compounds in the fluid sample are diverted into an evaporative light scattering detector. The evaporative light scattering detector generates a chromatographic signal of the sequentially eluted isolated compounds which is proportional to the total masses of the various compounds passing therethrough. An advantage of evaporative light scattering detection is that it is mass dependent and will tend to be generally uniform over a wide range of different chemical structures. Accordingly, a single calibration curve can be generated between masses passing through the evaporative light scattering detector and its signal output.
As stated, the mass spectrometer is used to selectively signal fraction collection of the compounds isolated in the fluid sample on the basis of their molecular weights and the light scattering detector signal is used to determine the actual total masses of the isolated compounds which are collected into the fraction collection microtiter plates.
In particular, the portion of each isolated compound diverted into the light scattering detector, (as compared to the remainder which is directed either to fraction collection or to waste), will be the same for all isolated compounds and will be determined by the dimensions of the splitter device used to divert a portion of the fluid sample into the light scattering detector. However, the portion of each isolated compound which is fraction collected as opposed to being diverted to waste will vary for different compounds, being dependent upon the portion of time during which fraction collection is carried out. The portion of time during which fraction collection is carried out will in turn depend upon the amount of time during which the compound is eluted at a sufficient purity for fraction collection.
By knowing the portion of each of the isolated compounds diverted into the light scattering detector as compared to the portion directed to fraction collection, and by knowing the actual mass passing through the light scattering detector, it is then possible to calculate the total masses of each of the individual fraction collected compounds. Specifically, the total mass of any particular isolated compound which is fraction collected is determined by measuring the signal generated by the light scattering detector, (which is proportional to the total mass of the compound passing to the fraction collector), during the interval in time in which the compound is eluted and fraction collected, (as determined by the mass spectrometer). By knowing the correlation between the signal generated by the light scattering detector and the amount of mass passing therethrough and by knowing the portion of fluid sample diverted into the light scattering detector as compared to the remainder of the fluid sample which passes directly to the fraction collector microtiter plate, the amount of mass present in any of the various isolated fraction collected compounds is calculated.
Initially, a calibration curve between the mass passing through the evaporative light scattering detector and the signal generated by the evaporative light scattering detector is determined. Such a calibration curve can be established by determining the signal output when passing known masses through the light scattering detector.
An additional advantage of the present invention is that as the actual masses of the fraction collected compounds are known when these compounds are fraction collected in microtiter plates, the microtiter plate fraction collector can then itself later be concentrated by being dried down. Afterwards, it can be reconstituted to set point molarity based upon the weight of each isolated compound as determined by the light scattering detector. As such, the microtiter plate can itself be used to create daughter plates directly without any reformatting or weighing.
Furthermore, by performing real time mass determinations of fraction collected samples, the present invention can be used to automatically calculate the yield of a given reaction. Moreover, such mass determinations can also be used as a control function when fraction collecting such that a maximum amount of an isolated compound can be collected, with the remainder being diverted to a second fraction collector or to waste.
In addition, the present system can be adapted to perform simultaneous real time mass determinations of a plurality of fraction collected samples, wherein a plurality of simultaneously eluted fluid samples can be analyzed by the evaporative light scattering detector and the mass spectrometer. In various aspects of the invention, parallel simultaneously eluted fluid samples pass through a switching valve such that each of the samples can be analyzed by the evaporative light scattering detector and the mass spectrometer in turn. In alternate aspects, parallel light scattering detection is performed by a plurality of lasers or with a single laser having its beam sequentially directed towards the various samples eluted in parallel.